The Protective Effect And Mechanism Of Notch-1Signaling Intervention On The High Glucose-induced Myocardial Cell Injury

Objective：1.To observe the effect of high glucose on H9c2myocardial cell proliferation,apoptosis and myocardial fibrosis.2.To observe the effect of lentiviral overexpressing Notch-1intervention onH9c2myocardial cell proliferation, apoptosis and myocardial fibrosis.3.To investigate the mechanisms of overexpress Notch-1attenuate the apoptosisand myocardial fibrosis of H9c2cells.Methods:1. The H9c2cells were cultured with in vitro with low sugar medium, and thento intervene by high sugar,γsecretion peptidase inhibitors (DAPT) and lentivirusrespectively.2. Six groups were divided:①Control group: The H9c2cells were cultured inlow sugar medium(5mmol/L);②Mannitol group:The H9c2cells were cultured in33mmol/L Mannitol medium;③High glucose group: The H9c2cells were culturedin33mmol/L glucose medium;④DAPT group: The H9c2cells were Intervented withspecific Notch-1blocker γ secretion peptidase inhibitors (DAPT);⑤Notch-1group:The H9c2cells were infected with lentiviral of Notch-1;⑥GFP group: The H9c2cells were infected with lentiviral of empty vector.3.48hours after DAPT intervention,72hours after lentivirus infection, theproliferation inhibition rate of H9c2cells was determined by CCK-8. ApplicateAnnexin V-FITC/PI measured by flow cytometry after double staining to detect theapoptotic rate in each group and compared. The mRNA expression of PI3K, AKTwere detected by qRT-PCR;The expression of Notch-1, p-PI3K/p-AKT, Bax, Bcl-2,TGF-β1, collagen I protein were detected by western blot.Results:1. The lentiviral vector of Notch-1overexpression was successfully constructed, the viral titer was2×108TU/ml, when the MOI is40, most viral were transfectedefficiency.2.The proliferation inhibition rate of each H9c2cardiomyocyte group wasdetected by CCK-8, the results showed that: High glucose can increase theproliferation inhibition rate of H9c2cardiomyocyte. Overexpression of Notch-1candecrease the proliferation inhibition rate of H9c2cardiomyocyte.The proliferationinhibition rate of H9c2cardiomyocyte was further increased after DAPT intervention,the difference was statistically significant (P <0.01).3. The expression of Bax, Bcl-2protein were detected by western blot, theresults showed that: High glucose can increase the atio of Bax/Bcl-2; Overexpressionof Notch-1can be reduced Bax/Bcl-2ratio, the ratio of Bax/Bcl-2was furtherincreased after DAPT intervention, the difference was statistically significant (P<0.05).4. Applicate Annexin V-FITC/PI measured by flow cytometry after doublestaining to detect the apoptotic rate in each group and compared, the results showedthat: high glucose can increase H9c2cardiomyocyte apoptosis rate, overexpression ofNotch-1can decrease H9c2myocardial apoptosis rate, apoptosis rate furtherincreased after the intervention of DAPT, the difference was statistically significant(P <0.05).5. The expression of TGF-β1, collagen I protein were detected by western blot,the results showed that: High glucose can upregulate the expression of TGF-β1andcollagen I protein, overexpression of Notch-1can downregulate the expression ofTGF-β1and collagen I protein, the expression of TGF-β1and collagen I proteinincreases up to a maximum value after the intervention of DAPT, the difference wasstatistically significant (P <0.05).6. The mRNA expression of PI3K/AKT was detected by qRT-PCR, the resultsshowed that: high glucose can downregulate The mRNA expression of PI3K/AKT,overexpression of Notch-1can upregulation PI3K/AKT mRNA, the expression ofH9c2cardiomyocytes PI3K/AKT mRNA is further decreased after the interventionof DAPT, the difference was statistically significant (P <0.05).7. The expression of notch-1、p-PI3K/p-AKT protein were detected by western blot, the results showed that: High glucose can downregulate the expression ofNotch-1, p-PI3K/p-AKT protein, overexpression of Notch-1can upregulate Notch-1,p-PI3K/p-AKT protein expression, Notch-1, p-PI3K/p-AKT protein expressionfurther downregulated after DAPT intervention, the difference was statisticallysignificant (P <0.05).Conclusions:1. The lentiviral vector of Notch-1overexpression was successfully constructedand infected H9c2cells effectively.2. High glucose can induce H9c2cardiac cells apoptosis and fibrosis reaction;overexpression of Notch-1can inhibit high glucose-induced apoptosis and fibrosisreaction.3. Overexpression of Notch-1can activate PI3K/AKT pathway, and AKT wasphosphorylated to regulate the expression of downstream, can also inhibit theexpression of TGF-β1, thereby downregulate the expression of collagen I, and mayinhibit high glucose-induced H9c2cardiac cells apoptosis and fibrosis reaction by thiseffect. Thus prompted that the integration role between PI3K/AKT pathway andNotch-1might be the main way of notch-1signal system reducing myocardialapoptosis and fibrosis reactions.